Methods of detecting gene expression in normal and cancerous cells

ABSTRACT

The present invention provides methods for detecting gene expression in normal and cancerous cells. Specifically, provided are methods utilizing molecular beacons (MB) technology combined with fluorescence imaging techniques for detecting, identifying or quantitating the presence of, or alterations in gene expression of, various tumor markers in a sample of cells.

This application is being filed on 13 Jan. 2004, as a PCT InternationalPatent application in the name of Emory University, a U.S. nationalUniversity, applicant for the designation of all countries except theUS, and Lily Yang, Gang Bo, Charles Staley, and Cynthia Cohen

FIELD OF THE INVENTION

This invention relates generally to methods of detecting human cancercells through examination of the levels of expression of tumor markergenes and mutant oncogenes in normal and/or cancerous cells usingmolecular beacon technology.

BACKGROUND OF THE INVENTION

Breast cancer is the most common type of cancer and a leading cause ofdeath among women. A crucial factor to increase survival is to diagnoseit early. Although early screening with mammography decreased themortality of the disease, nearly 20% of breast cancer patients are stillmissed by mammography. Furthermore, of all patients with abnormalmammograms, only 10 to 20% were confirmed to be breast cancer by biopsy(Harris et al., In Detita VT, Lippincott-Raven; 1557-1616 (1997)). Atpresent, there is no reliable serum tumor marker for diagnosis of breastcancer. Therefore, development of novel approaches for early diagnosisof breast cancer is of critical importance for the successful treatmentand for increasing survival of the patients.

At present, ductal lavage has been used as a minimally invasiveprocedure to collect breast ductal epithelial cells for cytopathologicalanalysis (O'Shaughnessy et al., Cancer 94(2):292-298 (2002)). Thisprocedure involves inserting a microcatheter into a nipple orifice,lavaging the cannulated duct with normal saline and collecting lavageeffluent. About 13,500 cells per duct can be collected for analysis ofthe presence of normal, atypical, or malignant breast ductal cells.However, the current method for identification of different cell typesis by morphological classification which is often inaccurate.

Pancreatic cancer is the fourth leading cause of cancer death in theUnited States because of its extremely poor prognosis (Parker et al.,CA. Cancer J. Clin. 46:5-27 (1996)). About 29,000 new cases arediagnosed and 28,000 of death occur each year in the United States (GoldE. I., Surg. Clin. North Am. 75:819-839 (1995)). Less than 50% ofpancreatic patients survive more than three month after diagnosis and 8%of them survive two years (National Institute of Health: NIH Publication93-2789 (1993)). The main reason for the poor prognosis is that very fewof the patients with pancreatic cancers are found early. Early diagnosisof pancreatic cancer using traditional radiographic and ultrasonographicmethods is extremely difficult (Barkin et al., Gastroenterology Clinicsof North America 28:709-722 (1999)). In spite of the extensivebiomedical research efforts during the last few decades, over 90% of thepatients with pancreatic cancer have already undergone local and/ordistant metastases by the time of diagnosis, often making it too late tocure. Therefore, it is extremely important to have early detection ofpancreatic cancer, possibly based on molecular markers rather than thesize of the tumor.

Molecular Markers of Pancreatic and Breast Cancer

It has been well established that currently K-ras oncogene is among themost attractive molecular markers for the detection of early pancreaticcancers (Minamoto et al., Cancer Detection & Prevention 24:1-12 (2000);Futakawa et al., Journal of Hepato-Biliary-Pancreatic Surgery 7:63-71(2000); Puig et al., International Journal of Cancer 85(1):73-77 (2000);Watanabe et al., Pancreas 17:341-7 (1998); Shibata et al., InternationalJournal of Oncology 12:1333-1338 (1998); Urgell et al., European Journalof Cancer 36:2069-2075, 2000)). A member of the G-protein family, K-rasis involved in signal transduction of growth-promoting effectors fromthe cell surface. Point mutations of K-ras are found in 80 to 100% ofpancreatic carcinomas, suggesting that it is a sensitive marker forcancer detection (Minamoto et al., Cancer Detection & Prevention 24:1-12(2000)). Further, most of these mutations are concentrated at codon 12,making K-ras even more attractive for the ease of beacon design. SinceK-ras mutations occur very early in the development of pancreaticcancer, tests targeting K-ras mutations can lead to early detection ofpancreatic carcinomas.

Recently, genes of a family of inhibitor of apoptosis proteins (IAPs)have been discovered (LaCasse et al., Oncogene 17:3247-3259 (1998)).IAPs inhibit the cascade of the apoptotic pathway through inhibition ofcaspase activity (Deveraux et al., EMBO Journal 17(8):2215-2223 (1998)).Increasing evidence indicate that one of the LAP proteins, survivin, isalso a promising tumor marker for several types of cancers (Altieri etal., Lab. Invest. 79:1327-1333 (1999); Tanaka et al., Clin. Cancer Res.6:127-134 (2000); Moore et al., J. of Insurance Med. 33:202-203 (2001);Altieri et al., Trends in Mol. Med. 7:542-547 (2001)). Survivin isnormally expressed during fetal development but is not expressed in mostnormal adult tissues (Altieri et al., Lab. Invest. 79:1327-1333 (1999)).Results from the analysis of 3.5 million transcripts from 19 humannormal and diseased tissue types have also revealed that survivin is oneof the top four genes that is uniformly expressed at elevated levels inall cancer tissues examined, but not in normal tissues (Velculescu etal., Nature Genetics 23:387-388 (1999)). A recent study has demonstratedthe presence of survivin in 77% of pancreatic duct cell adenocarcinomaand 56% intraductal papillary-mucinous tumor (IPMT) byimmunohistochemistry, immunoblotting and RT-PCR assays (Saton et al.,Cancer 92:271-278 (2001)). Expression of survivin can be detected in allstages (from I to IV) of pancreatic duct cell carcinoma including inearly stages of neoplastic transition in pancreatic cancer cells.However, expression of survivin was not detected in normal pancreatictissues, inflammatory cells around tumor cells and pancreatic tissuesfrom patients with chronic pancreatitis. Absence of survivin expressionin normal pancreas and other normal tissues makes it an ideal molecularmarker for the detection of pancreatic cancer cells.

The transition from normal mammary epithelial to invasive ductalcarcinoma is a multistage process, which involves a series ofhistological changes in the breast tissues from hyperplasia, atypicalhyperplasia to duct carcinoma in situ (DCIS) and to invasive ductalcarcinoma. Several tumor markers have been found to be present in DCISlesions and invasive breast cancers. Cyclin D 1, an important regulatorfor cell cycle, is overexpressed in 80% of DCIS whereas it is low orabsent in normal breast tissues (Weinstat-Saslow et al., Nat Med1(12):1257-1260 (1995); Vos et al., J. of Path. 187(3):279-84 (1999)).Amplification and overexpression of Her-2/neu gene are also demonstratedin 30% of invasive breast cancers and 60 to 80% of DCIS tissues (Janockoet al., Cytometry 46(3):136-49 (2001); Poller et al., Breast Cancer Res.& Treat. 20(1):3-10(1991); Ramachandra et al., J. of Path. 161(1):7-14(1990)). However, overexpression of Her-2/neu is not found in normalductal cells and in simple hyperplasia (Poller et al., Breast CancerRes. & Treat. 20(1):3-10 (1991); Ramachandra et al., J. of Path.161(1):7-14 (1990)). This suggests that regulation of these genes maydefine a transition from a benign state to carcinoma and thatunregulated overexpression of cyclin D 1 and Her-2/neu may be a commonearly event in mammary carcinomas. Additionally, high levels of survivinare also detected in 71% of breast cancer tissues obtained from thepatients with invasive and metastatic breast cancers while thesurrounding normal breast tissues are negative. An increase in thelevels of survivin contributes to a higher apoptotic threshold andsurvival ability of the breast tumor cells (Tanaka et al., Clin. CancerRes. 6:127-134 (2000)). Expression of survivin gene appears to be anearly tumor marker in developing breast cancer since a high level ofsurvivin is detected in over 80% of DCIS tissues of the breast cancerpatients (Yang L. et al. Unpublished observations). Therefore, cyclin D1, Her-2/neu and survivin are sensitive tumor markers for earlydetection of breast cancer cells at pre-invasive stage.

Survivin is also detected in many common tumor types such as prostate,lung, colon, gastric, liver, brain, renal, melanoma and lymphoma(Altieri et al., Trends in Mol. Med. 7:542-547 (2001)). For example, 64%of human colorectal cancers express a high level of survivin. Five-yearsurvival rate in the stage II patients with positive survivin are muchless than that of the survivin negative patients (Sarela et al., Gut46(5):645-650 (2000)). The correlation of the survivin expression andprognosis of cancer patients has also been demonstrated in several othertumor types (Kappler et al., International Journal of Cancer. 95:360-363(2001); Swana et al., New England Journal of Medicine. 341:452-453(1999)). Several reports indicated that the expression of survivin genein tumor cells contributed to resistance of the tumor cells to chemo- orradiotherapy (Kato et al., International Journal of Cancer 95:92-5(2001); Azuhata et al., Journal of Pediatric Surgery 36:1785-1791(2001); Asanuma et al., Japanese Journal of Cancer Research 91:1204-9(2000)). Therefore, level of survivin gene expression could be used todetermine the sensitivity of the human tumor cells to chemotherapydrugs.

Detecting Human Cancer Cells with the Molecular Beacon Technology

At present, the commonly used methods for detection of the gene mutationin clinical samples are DNA purification of genomic DNA or RNA isolationfollowed by mutant-enriched PCR or RT-PCR. The presence of mutant PCRproducts is then determined by single strand conformation polymorphism(SSCP), restriction fragment-length polymorphisms (RELP), orallele-specific oligodeoxynucleotide hybridization (ASOH) (Futakawa etal., Journal of Hepato-Biliary-Pancreatic Surgery 7:63-71 (2000); Puiget al., International Journal of Cancer 85(1):73-77 (2000); Watanabe etal., Pancreas 17:341-7 (1998); Shibata et al., International Journal ofOncology 12:1333-1338 (1998); Fischer et al., Laboratory Investigation81:827-831 (2001); Clayton et al., Clinical Chemistry 46:1929-1938(2000)). Although identification of K-ras mutations by PCR is a fairlysensitive molecular approach, the procedures for PCR and subsequentassays are very time-consuming, making them difficult to become clinicalprocedures. So far, it has been very difficult to directly detect theexpression of mutant oncogenes in intact cells since in situhybridization method with current fluorescence-labeled-linearoligonucleotide probes have a high level of fluorescence background anda low sensitivity in detecting mRNAs with single base pair mutation.Immunostaining with antibodies to mutant proteins usually lacksspecificity and generates “false positive” data A high number of “falsepositives” has been observed due to non-specific labeling and presenceof endogenous peroxidase or alkaline phosphatase. Thus, it is importantto develop more specific assays for detection of the tumor cellsexpressing a mutant oncogene.

It is well established that cancer cells develop due to geneticalterations in oncogenes and tumor suppressor genes and abnormalities ingene expression that provide growth advantage and metastatic potentialto the cells. A hereunto for utilized method of achieving earlydetection of cancer would be to identify the cancer cells throughdetection of mRNA transcripts that are expressed in the cancer cells butis low or not expressed in normal cells. Therefore, aheretofore-unaddressed need exists in the art to address theaforementioned deficiencies and inadequacies.

SUMMARY OF THE INVENTION

Various aspects of the present invention provide methods for detectingthe level of gene expression in fixed or viable normal and cancerouscells. Specifically, provided are methods utilizing molecular beacons(MB) technology combined with fluorescence imaging techniques fordetecting, identifying or quantitating the presence of, or alterationsin gene expression of, various tumor markers in a sample of cells.

MBs are single-stranded oligonucleotides with a fluorophore at one endand a quencher at the other; they are designed to form a stem-loopstructure when their target mRNA is not present such that thefluorescence of the fluorophore is quenched. The loop portion has aprobe sequence complementary to a target mRNA molecule. The armsequences near each end of the loop are complementary to each other;they anneal to form the MB's stem. When the MB encounters a target mRNAmolecule, the loop and possibly a part of the stem hybridize to thetarget mRNA, causing a spontaneous conformational change that forces thestem apart. The quencher moves away from the fluorophore, leading to therestoration of fluorescence. A major advantage of the stem-loop probesis that they can recognize their targets with a higher specificity thanthe linear probes. Properly designed MBs could discriminate betweentargets that differ by as little as a single nucleotide. The design ofMBs also allows specific binding of the MBs to their target nucleotidesequences and reports the hybridization through generating afluorescence signal without separation of unbound probes from MB-targetcomplex since free MBs do not fluoresce. Therefore, MBs should provideus with an excellent tool for detecting specific nucleotide sequence,such as mRNA and DNA, with a high noise to signal ratio in intact cellsas well as in solution.

Thus, in one aspect, the invention is related to a method of detectingthe presence of at least one tumor marker mRNA in a sample. The methodincludes providing a sample of cells for analysis and then treating thesample with a circle oligonucleotide (MB) that targets the tumor markermRNA. The hybridization of the target sequence is then detected,identified or quantitated under suitable hybridization conditions, suchthat the presence, absence or amount of target sequence present in thesample is correlated with a change in detectable fluorescence signal.The presence of a tumor marker can then be detected, identified orquantitated based upon the presence, absence or amount of thehybridization of the oligonucleotide to the target sequence that isdetermined. The MBs can be delivered into acetone-fixed cells by directincubation or into viable cells through transfection. The presence andquantification of level of tumor marker mRNAs after delivery of the MBsinto fixed or viable tumor cells are accomplished by measuring thefluorescence intensity using a fluorescence microscope, using FACS-scananalysis of individual cell populations or monitoring the changes of therelative fluorescence unit real-time in 96-well plate using afluorescence microplate-reader.

The tumor marker to be detected and be any tumor marker and in certainaspects of the present invention can include one or more of thefollowing: survivin, cyclin D1, Her2/neu, a mutant K-ras,chymotrypsinogen, XIAP, basic fiborblast growth factor, EGF receptor,carcinoembryonic antigen, prostate, specific antigen, alpha-fetoprotein,beta-2 microglobulin, bladder tumor antigen, chromogranin A,neuron-specific enolase, S-100, TA-90, tissue polypeptide antigen andhuman chorionic gonadotropin.

In any aspect of the present invention, the sample can be taken from oneor more of any number of sources including, but not limited to, blood,urine, pancreatic juice, ascites, breast ductal lavage, nippleaspiration, needle biopsy or tissue. In certain aspects of theinvention, the tissue is a biopsy from the pancreas or breast. In otheraspects of the invention, the tissue can be in the form of a frozenmicroscope section.

Another aspect of the present invention related to a method fordetecting the presence of a mutant gene in a tumor cell that includesproviding a sample of tumor cells for analysis and then treating thesample with an oligonucleotide that targets the mutant gene. In certainaspects of the present invention, the mutant gene is a mutant K-rasgene.

Still another aspect of the present invention relates to a method ofmonitoring the level of gene expression in viable cells.

Yet another aspect of the invention relates to a method of detecting ormonitoring the presence or progression of breast cancer in a subjectthat includes monitoring or detecting the presence of a breast cancermarker. In various aspects of the invention, the breast cancer markercan be one or more of the following: survivin, cyclin D1, Her2/neu,basic fibroblast growth factor and carcinoembryonic antigen. In certainaspects of the invention, the sample can be taken from, but is notlimited to, one or more of the following: blood, urine, breast ductallavage, nipple aspiration, ascites needle biopsy or tissue.

Still yet another aspect of the present invention relates to a method ofdetecting or monitoring presence or progression of pancreatic cancer ina subject that includes detecting or monitoring for the presence of apancreatic cancer marker. In various aspects of the invention, thepancreatic cancer marker can be, but is not limited to, one or more of:survivin, a mutant K-ras gene, and carcinoembryonic antigen. Variousaspects of the invention also provide that the sample can be taken fromat least one source including, but not limited to, blood, urine,pancreatic juice, needle biopsy or tissue.

Another aspect of the present invention relates to a method of detectingcancerous cells in a sample that includes treating a sample of cellswith an oligonucleotide that targets a cancer-specific marker genesequence. In various aspects of the invention, the cancer cell canoriginate from one or more of the following cancers, including but notlimited to, breast, pancreas, ovarian, prostate, colorectal,hepatocellular, multiple myeloma, lymphoma, bladder, medullary carcinomaof the thyroid, neuroendocrine tumors, carcinoid tumors, testicular,gestational trophoblast neoplasms, lung, melanoma and stomach.

Other aspects of the present invention provide diagnostic kits for 1)detecting or monitoring the progression of cancerous cells; and, 2)detecting the level of gene expression in viable cells in real-time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic illustration of molecular beacons (MBs). (A)Molecular beacons are dual labeled oligonucleotides with a hairpinstructure; (B) delivering molecular beacons into cells can result in afluorescent signal due to hybridization of the probe with a target mRNA.

FIG. 2 shows specific binding of molecular beacon probes to theiroligonucleotide targets in vitro. K-ras MB1, K-ras MB2, survivin andcyclin D1 MBs were mixed with synthesized specific or non-specific DNAtargets and incubated for 1 hour at 37° C. (survivin and cyclin D1) or50° C. (K ras). The relative fluorescence unit (RFU) was measured in afluorescence microplate reader. The bar in the figure is the mean RFU offour repeat samples. FIGS. 2A and B: K-ras MB 1 or MB 2 specificallybound to K-ras Mut 1 or Mut 2 target resulting in a higher fluorescenceintensity as compared with K-ras WT target or the target with adifferent k-ras mutation. FIGS. 2C and D. Survivin MB or cyclin D1 MBhybridized only to survivin or cyclin D1 target but do not bind to K-rasor Her-2 target resulting in a high level of fluorescence signal.

FIG. 3 depicts the molecular beacon imaging of pancreatic cancer cellsby simultaneous detection of expression of mutant K-ras and survivingenes in human Pancreatic cancer cell lines. Pancreatic cancer andnormal cell lines were fixed with acetone. The cells were incubated withmixtures of K-ras MB 1-cy 3 (GGT to GAT) with survivin MB-FITC or K-rasMB2-Texas red (GGT to GTT) with survivin MB-FITC for 1 hour at 50° C.The slides were then observed under a confocal fluorescence microscope.The fluorescent images were taken and same exposure condition was usedto take all images for each color. The cells with red fluorescence werepancreatic cancer cells expressing specific mutant K-ras as detectedeither by K-ras MB 1 or K-ras MB 2. The cells were also double labeledwith green fluorescence showing that these tumor cells also expressed atumor marker survivin. FIG. 3A shows the specificity of detection ofpancreatic cancer cells expressing mutant K-ras and survivin genes inthe tumor cell lines with specific K-ras mutations and a high level ofsurvivin. Panc-1 cell line has a K-ras GGT to GAT mutation and showedstrong fluorescence intensity after incubating with K-ras MB 1, butdisplayed a weak fluorescence in K-ras MB2-stained cells. Capan-2 cellline contains a K-ras GGT to GTT mutation and a brighter fluorescencewas detected in K-ras MB 2 stained cells. Both cell lines expressed ahigh level of survivin as detected by survivin MB. FIG. 3B showspancreatic cancer cells expressing a different mutant or wild type K-rasgene showed a weak or negative for K-ras MBs but can still be detectedby survivin MB. K-ras MBs did not produce strong fluorescence signalingin MIA PaCa-2 cell line, which has a K-ras GGT to TGT mutation, or K-raswild type BXPC-3 cells. However, those cells were positive for survivinMB staining. Incubation of K-ras and survivin MBs did not producedetectable fluorescence signaling in a normal cell line (HDF), which isgenerated from normal dermal fibroblasts. FIG. 3C shows the comparisonof fluorescence intensity of pancreatic cancer and normal cells afterdelivery of K-ras MBs. The fluorescent intensity was measured in eachimage at three randomly selected areas using Adobe Photo Shop software.The numbers in the bar figure represent the mean fluorescence intensityof 5 to 7 images.

FIG. 4 depicts molecular beacon imaging of human breast cancer cellsexpressing tumor markers cyclin d1 and survivin. FIG. 4A shows breastcancer and normal mammary epithelial lines that were incubated with amixture of cyclin D 1-texas red and survivin MB-Alexa 488 for 1 hour at37° C. The slides were then observed under a confocal fluorescencemicroscope. The fluorescent images were taken and same exposurecondition was used to take all images for each color. FIG. 4B showslevels of fluorescence intensity in breast cancer cell lines detected bysurvivin and cyclin D1 MBs are correlated the levels of survivin andcyclin D1 proteins detected by Western blot analysis.

FIG. 5 shows the detection of survivin expression on frozen sections ofhuman breast cancer tissues at DCIS and invasive stages byimmunofluorescence staining for survivin protein or survivin mbdetecting survivin mRNA. Frozen tissue sections of breast cancer ornormal tissues were incubated with either a polyclonal survivin antibodyor survivin MB-cy3. FIG. 5A. Survivin is early tumor marker and bothsurvivin mRNA and protein could be detected in the early stage of breastcancer, duct carcinoma in situ (DCIS). Survivin is also detected ininvasive breast cancer but not in normal breast tissues. FIG. 5B.Survivin MB detected metastatic breast cancer cells in the lymph node,indicating the feasibility of development of a quick and sensitivitymethod for detecting the presence of breast cancer cells in the lymphnode of the patients.

FIG. 6 depicts specific imaging of pancreatic cancer cells expressingmutant K-ras and survivin mRNAs on frozen tissue sections of pancreaticcancer tissues. Frozen tissue sections were incubated with K-ras MB 1 orK-ras MB 2, or survivin MB for 1 hour and counterstained with Hoechst33342 (blue) FIG. 6A: K-ras MB 1 detected the cancer cells expressing aGGT to GAT mutant K-ras gene on the frozen sections of pancreatic cancertissues from patient #1 and #2, which contained K-ras codon 12 GGT toGAT mutation. However, bright red fluorescent cells were found on frozensections of pancreatic cancer tissues from patient #5, which had a K-rasGGT to GTT mutation after incubation with K-ras MB 2 but not with K-rasMB1. FIG. 6B: Detection of the levels of survivin protein or mRNA inpancreatic cancer cells on frozen tissue sections by immunofluorescenceor survivin MB staining. For detection of the levels of survivin proteinby immunofluorescence staining, frozen tissue sections of pancreaticcancer and normal tissues were incubated with a mouse anti-survivinantibody (survivin Ab) followed by a FITC-labeled goat anti-mousesecondary antibody. High levels of survivin protein and mRNA were foundin pancreatic cancer tissues but not in the normal pancreatic tissues.

FIG. 7 shows the real-time detection of survivin gene expression inviable breast cancer cells after EGF treatment. FIG. 7A: breast cancercell line MCF-7 was cultured in 96-well plate and placed in the mediumcontaining 2% FBS for overnight. The cells were then transfected with amixture of 200 nM of survivin-6 FAM and GAPDH-cy3 MBs usinglipofectamine 2000 for three hours. 200 ng of EGF was then added to themedium. The tissue culture plate was placed immediately in afluorescence microplate reader and the relative fluorescence unit (RFU)was measured every 30 minutes for three hours. The curve in the figurerepresents the mean number of three repeat samples and is a ratio of RFUof 6-FAM (survivin MB) and cy3 (internal control GAPDH MB). FIG. 7B: Thetransfected cells treated with or without EGF were observed at 24 hrsunder a confocal microscope and the fluorescence images were taken.

FIG. 8 shows the real-time detection of changes of survivin geneexpression using survivin MB at different time points followingdocetaxel treatment. FIG. 8A: Human breast cancer cell lines MCF-7 andMDA-MB-231 were cultured in 96 well plate. After transfecting with 100nM of survivin and GAPDH MB mixture, the cells were treated with orwithout chemotherapy drug docetaxel and the changes of fluorescenceintensity in each group were measured real time in a fluorescencemicroplate reader for 48 hours. FIG. 8B: The transfected cells treatedwith or without 20 (MDA-MB-231) or 50 nm (MCF-7) of docetaxel for 24 to48 hrs and then observed under a confocal microscope and thefluorescence images were taken.

FIG. 9 depicts the level of survivin gene expression in viable cellscould be detected by FACscan analysis. Breast cancer cell lineMDA-MB-231 and normal cell line HDF were transfected with survivin orβ-actin MB for 3 hours. The cells were collected from culture dishes andanalyzed with FACScan for the level of survivin gene expression innormal and tumor cells. The result demonstrated that the level of geneexpression in viable cells could also be measured quantitatively usingFACScan.

FIG. 10 shows the detection of the levels of survivin in breast normaland cancer tissues by Western blot analysis. Levels of survivin inpaired or non-paired normal and cancer tissue samples from the breastcancer patients.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Various embodiments of the invention are now described in detail. Asused in the description herein and throughout the claims that follow,the meaning of “a,”“an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein and throughout the claims that follow, the meaning of “in”includes “in” and “on” unless the context clearly dictates otherwise.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitioner indescribing the compositions and methods of the invention and how to makeand use them. For convenience, certain terms may be highlighted, forexample using italics and/or quotation marks. The use of highlightinghas no influence on the scope and meaning of a term; the scope andmeaning of a term is the same, in the same context, whether or not it ishighlighted. It will be appreciated that the same thing can be said inmore than one way. Consequently, alternative language and synonyms maybe used for any one or more of the terms discussed herein, nor is anyspecial significance to be placed upon whether or not a term iselaborated or discussed herein. Synonyms for certain terms are provided.A recital of one or more synonyms does not exclude the use of othersynonyms. The use of examples anywhere in this specification, includingexamples of any terms discussed herein, is illustrative only, and in noway limits the scope and meaning of the invention or of any exemplifiedterm. Likewise, the invention is not limited to various embodimentsgiven in this specification.

As used herein, “about” or “approximately” shall generally mean within20 percent, preferably within 10 percent, and more preferably within 5percent of a given value or range. Numerical quantities given herein areapproximate, meaning that the term “about” or “approximately” can beinferred if not expressly stated.

Definitions

“Hybridization” and “complementary” as used herein, refer to thecapacity for precise pairing between two nucleotides. For example, if anucleotide at a certain position of an oligonucleotide is capable ofhydrogen bonding with a nucleotide at the same position of a DNA or RNAmolecule, then the oligonucleotide and the DNA or RNA are considered tobe complementary or hybridizable to each other at that position. Theoligonucleotide and the DNA or RNA hybridize when a sufficient number ofcorresponding positions in each molecule are occupied by nucleotideswhich can hydrogen bond with each other. It is understood in the artthat the sequence of an antisense oligonucleotide need not be 100%complementary to that of its target nucleic acid to hybridize thereto.An oligonucleotide is specifically hybridizable when binding of thecompound to the target DNA or RNA molecule, and there is a sufficientdegree of complementarity to avoid non-specific binding of the antisenseoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, e.g., under physiological conditions in thecase of in vivo assays or therapeutic treatment, or, in the case of invitro assays, under conditions in which the assays are performed.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes, but is not limitedto, oligonucleotides composed of naturally occurring and/or syntheticnucleobases, sugars, and covalent internucleoside (backbone) linkages.Such modified or substituted oligonucleotides are often preferred overnative forms because of desirable properties such as, for example,enhanced cellular uptake, enhanced affinity for nucleic acid targets,and/or increased stability in the presence of nucleases.

The present invention provides methods for detecting gene expression innormal and cancerous cells. Specifically, provided are methods fordetecting, identifying or quantitating the presence of, or alterationsin gene expression of, various tumor markers in a sample of cells.

Inventors have developed a molecular beacon (MB) technology to detectgene expression in viable as well as fixed tumor cells. MBs areoligonucleotides with a stem-loop hairpin structure, dual-labeled with afluorophore at one end and a quencher at the other. Delivering MBs intocells will result in a fluorescence signal if the MBs hybridize totarget mRNAs. Thus, when the target mRNAs correspond to the molecularmarkers of a cancer, cancer cells (bright) can be distinguished fromnormal cells (dark). Methods are provided for: detecting the presence ofat least one tumor marker mRNA in a sample of cells; detecting thepresence of a mutant gene in a tumor cell; monitoring alterations ingene expression in viable cells; detecting or monitoring the presence orprogression of breast cancer in a subject that includes monitoring ordetecting the presence of a breast cancer marker; detecting ormonitoring presence or progression of pancreatic cancer in a subjectthat includes detecting or monitoring for the presence of a pancreaticcancer marker; and, detecting cancerous cells in a sample that includestreating a sample of cells with an oligonucleotide that targets acancer-specific marker gene sequence. Diagnostic kits are also providedthat 1) detect or monitor the progression of cancerous cells; and, 2)detect alterations in gene expression in viable cells in real-time.

Molecular Beacons

As outlined in FIG. 1, MBs are single-stranded oligonucleotides with afluorophore at one end and a quencher at the other. Although by no meanslimiting, typically, the fluorophore is attached to the 5′ end while thequencher is attached to the 3′ end. They are designed to form astem-loop structure when their target mRNAs are not present such thatthe fluorescence of the fluorophore is quenched. The loop portion has aprobe sequence complementary to a target mRNA molecule. Typicalfluorophores that are contemplated to be used include, but are notlimited to, Cy3 fluorophore (Cy3 Amidite, Amersham Pharmacia Biotech,Piscataway, N.J.) Alexa Fluor 488 (Molecular Probes); Alexa Fluor 350(blue), CMAC (7-amino-4-chloromethylcoumarin), 6-FAM and FITC. Typicalquenchers include, but are not limited to,dabcyl(4-(4′-dimethylaminophenylazo)benzoic acid) (Dabcyl-CPG, GlenResearch, Sterling, Va.).

When the MB encounters a target mRNA molecule, the loop and a part ofthe stem hybridize to the target mRNA, causing a spontaneousconformational change that forces the stem apart. The quencher movesaway from the fluorophore, leading to the restoration of fluorescence(Tyagi et al., Nature Biotechnol 14:303-308 (1996); Dubertret et al.,Nat Biotechnol 19:365-370 (2001)). One major advantage of the stem-loopprobes is that they can recognize their targets with a higherspecificity than the linear oligonucleotide probes. Properly designedMBs can discriminate between targets that differ by as little as asingle nucleotide (Tyagi et al., Nat Biotechnol 16:49-53 (1998)). TheMBs have been utilized in a variety of applications including DNAmutation detection, protein-DNA interactions, real-time monitoring ofPCR, gene typing and mRNA detection in living cells (Dubertret et al.,Nat Biotechnol 19:365-370 (2001); Dirks et al., Histochem. & Cell Biol.115(1):3-11 (2001); Tyagi et al., Nat Biotechnol 16:49-53 (1998); Sokolet al., Proc Natl Acad Sci USA 95:11538-11543 (1998)).

The various embodiments of the present invention provide methods for:detecting the presence of at least one tumor marker mRNA in a sample ofcells; detecting the presence of a mutant gene in a tumor cell;monitoring alterations in gene expression in viable cells; detecting ormonitoring the presence or progression of breast cancer in a subjectthat includes monitoring or detecting the presence of a breast cancermarker; detecting or monitoring presence or progression of pancreaticcancer in a subject that includes detecting or monitoring for thepresence of a pancreatic cancer marker; and, detecting cancerous cellsin a sample that includes treating a sample of cells with anoligonucleotide that targets a cancer-specific marker gene sequence,such methods comprising:

-   -   i) providing a sample of cells for analysis;    -   ii) treating the sample with an oligonucleotide that targets the        desired marker or gene;    -   iii) detecting, identifying or quantitating the hybridization of        the target sequence under suitable hybridization conditions,        wherein the presence, absence or amount of target sequence        present in the sample is correlated with a change in detectable        signal associated with at least one donor or acceptor moiety of        the oligonucleotide; and    -   iv) detecting, identifying or quantitating the presence of a        marker or gene based upon the presence, absence or amount of the        hybridization of the oligonucleotide to the target sequence that        is determined.

It is contemplated by the inventors that any oligonucletode constructedfor the use as a molecular beacon as described above can be used inmethods of the present invention. In particular embodiments, theolignucleotide includes, but is not limited to, one or more of SEQ IDNOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In one embodiment,the oligonucleotide targets the tumor marker survivin. In a preferredembodiment, the oligonucleotide that targets survivin includes, but isnot limited to, one or more of SEQ ID NOS: 1, 2, and 9. In anotherembodiment, the oligonucleotide targets the tumor marker cyclin D1. In apreferred embodiment, the oligonucleotide that targets cyclin D1,includes, but is not limited to, one or of SEQ ID NOS: 3 and 4. Inanother embodiment, the oligonucleotide targets the tumor markerHer2/neu. In a preferred embodiment, the oligonucleotide that targetsHer2/neu includes, but is not limited to, one or more of SEQ ID NOS: 5and 6. In yet another embodiment, the oligonucleotide targets the K-rasmutant gene tumor marker. In a preferred embodiment, the oligonucleotidethat targets the K-ras mutant gene includes, but is not limited to, oneor more of SEQ ID NOS: 7, 8, 11, 12 and 13.

Detection of Tumor Markers

In various embodiments, the present invention provides a method ofdetecting the presence of at least one tumor marker mRNA in a sampleusing the molecular beacon technology described herein. As one soskilled in the art will readily appreciate, the methods of the presentinvention can be utilized to detect the presence of any tumor markermRNA present in a sample. Such markers include but are not limited to,survivin, cyclin D1, Her2/neu, a mutant K-ras, basic fibroblast growthfactor, EGF receptor, XIAP, carcinoembryonic antigen, prostate, specificantigen, alpha-fetoprotein, beta-2-microglobulin, bladder tumor antigen,chromogranin A, neuron-specific enolase, S-100, TA-90, tissuepolypeptide antigen and human chorionic gonadotropin.

Sample Collection: Samples of cells to be tested can be obtained throughroutine diagnostic procedures. Such samples can include, but are notlimited to, blood, urine, fine needle aspirates, breast ductal lavage,pancreatic juice, ascites, nipple aspiration samples, or any othertissue, including, but not limited to, a biopsy from anywhere from thepatient, including, but not limited to, the breast, the pancreas or alymph node. The tissue sample can be any type of routine pathologicallyprepared sample including any type of tissue affixed to a microscopeslide, plate or well. In a preferred embodiment, the tissue sample is afrozen section of tissue. In another preferred embodiment, the sample istaken from a breast ductal lavage. In still another preferredembodiment, the sample is taken from pancreatic juice.

Detection of Breast Cancer Tumor Markers

Certain embodiments of the present invention provide a method ofdetecting or monitoring the presence or progression of breast cancer ina subject using the molecular beacon technology described herein. Thepresent invention provides methods for detecting breast cancer cellsfrom ductal lavage and fine needle aspiration (FNA) using a combinationof MBs targeting the mRNAs of genes that have been shown to be expressedin the early stage of tumorigenesis in breast cancer. The inventors haveshown the predictive values of the detection of each gene or monitoringthe co-expression of two or three genes in the diagnosis of ductalcarcinoma in situ. In certain embodiments of the present invention,methods are provided that simultaneously detect of the overexpression ofsurvivin, cyclin D 1 and Her-2/neu genes in breast ductal epithelialcells. The methods of the invention are useful to detect the presence ofthese tumor markers especially when a tumor single cell expresses morethan one marker gene. In a preferred embodiment, a method is providedfor the detection of survivin, cyclin D1 and/or Her-2/neu expressingcells in the ductal lavage.

In various embodiments, the methods utilize MBs designed to specificallyhybridize to mRNAs of survivin, cyclin D 1 or Her-2/neu. Applicants havedemonstrated the specificity and sensitivity of the MBs in human breastcancer cell lines, normal mammary epithelial and normal fibroblast celllines as well as in identifying the isolated tumor cells in a backgroundof normal cells with different cancer- to normal-cell ratios. Theprovided methods can be utilized to detect the expression of these tumormarkers in the cellular fractions of ductal lavage and aspirates of fineneedle aspirates obtained from early stage breast cancer or ductalcarcinoma in situ (DCIS) patients at different stages of the disease andnormal control subjects.

Sample Collection: Samples of cells to be tested can be obtained throughroutine diagnostic procedures. Such samples can include, but are notlimited to, blood, urine, fine needle aspirates, breast ductal lavage,ascites, nipple aspiration samples, or any other tissue, including, butnot limited to, a biopsy from anywhere on the breast or a lymph node.The tissue sample can be any type of routine pathologically preparedsample including any type of tissue affixed to a microscope slide, plateor well. In a preferred embodiment, the tissue sample is a frozensection of tissue.

It is contemplated that samples can be obtained through any routinediagnostic procedure for breast cancer patients or women at a high riskfor developing breast cancer. As a non-limiting example, a ductal lavagefrom a cancer patient is collected when the patient is undergoingsurgery to remove the breast cancer or in a routine visit to doctor'soffice. Under anesthesia, a microcathether is inserted into the duct andsaline infused. The effluent fluid is collected and cellular fractionenriched by centrifugation. The enriched cell fraction from ductallavage or aspirates are then placed on glass slides. Typically, about 10to 15 cytospin slides are obtained from one ductal lavage with a medianof 13,500 epithelial cells per duct. After fixing the cells in ice-coldacetone, the slides are incubated with one or more MBs, eithersequentially or simultaneously, at optimized incubation conditions andthen examined under a fluorescence microscope. Since MBs for each geneare labeled with different fluorescent dyes, the number of the cellsover-expressing any or all of the genes for the target tumor markers ina sample are determined.

Analysis: The inventors contemplate that results obtained from one typeof sample collection can be compared with those obtained from another inorder to aid in the identification, monitoring or detection of a tumormarker or in the diagnosis of, or monitoring the progression of thecancer. Subsequently, the same slides can be stained and analyzed by acytopathologist for the presence of benign, atypical or malignant cells.Such staining can include routing cytological stains such as H&E orimmuostaining with specific antibodies.

Quantification: Detection of the hybridization of an MB with its targetsequence in intact cells, either fixed or viable, can be accomplished byfluorescence microscopy, FACS analysis and fluorescence microplatereader. As a non-limiting example, the breast cancer cells wereco-transfected with survivin and GAPDH MBs and then treated with EGF.The alternation of survivin gene expression after EGF treatment wasmonitored real time in microplate reader for 3 hours. The results showedthat EGF induced an increase in survivin gene expression within 30minutes of the treatment.

Detection of Pancreatic Cancer Tumor Markers

Certain embodiments of the present invention provide a method ofdetecting or monitoring the presence or progression of pancreatic cancerin a subject using the molecular beacon technology described herein.

One of the crucial issues for early detection of pancreatic cancer is todevelop assays that are capable of identifying a few tumor cells in apool of a large number of normal cells. At present, RT-PCR is the mostsensitive assay for detection of the genes that are highly expressed intumor cells or for mutated gene products such as mutant K-ras gene, orcarcinoembryonic antigen. Though RT-PCR can detect one tumor cell in 10⁴to 10⁵ cells, such assays may generate ‘false positives’. In addition,using current molecular markers, RT-PCR detection of gene expression ormutant gene in peripheral blood and pancreatic juice cannot localize thecancer to pancreas since many types of cancers as well as othernon-malignant diseases may also express those molecular markers.Further, RT-PCR assays are very time consuming, typically detecting onegene at a time, making it difficult to become an efficient clinicalprocedure for cancer diagnosis.

The inventors have developed a sensitive and more efficient method asdisclosed herein that can identify a small number of pancreatic cancercells in peripheral blood and pancreatic juice samples. The MB-basedmethods disclosed herein can be used to detect pancreatic cancer ortumor cells from a mixed cell population using a single MB type or acombination of several MBs. The methods provided can be used to detectK-ras mutations after RT-PCR amplification of K-ras exon 1. In addition,various cytological or immunostaining procedures can be used inconjunction with the disclosed methods.

For early detection of pancreatic cancer in the high-risk patientpopulation or patients suspected to have pancreatic cancer, it isimportant to develop clinical assays from patient samples that can beobtained non-invasively or by a minimally invasive procedure. Increasingevidence has revealed the presence of disseminated tumor cells in blood,bone marrow and peritoneal cavity of pancreatic cancer patients (LaCasseet al., Oncogene 17:3247-3259 (1998); Li et al., Nature 396:580-584(1998); Tamm et al., Cancer Research 58:5315-5320 (1998); Ambrosini etal., Nature Medicine 3:917-921 (1997)). For example, byimmunohistochemical staining using antibodies detecting severalcytokeratin and tumor markers, the cancer cells were found in 28% of theblood samples obtained from patients with pancreatic cancers. Theprevalence of finding cancer cells in blood samples increased with tumorstage. However, it failed to find cancer cells in the blood samples ofstage 1 pancreatic cancer patients (Z'graggen et al., Surgery129:537-545 (2001)). The present invention provides sensitive methods inwhich various samples from pancreatic cancer patients of all stages canbe examined using the MB technology disclosed herein to identify cellsexpressing pancreatic tumor markers including, but not limited to,mutant K-ras and survivin genes.

Sample Collection: Samples of cells to be tested can be obtained throughroutine diagnostic procedures. Such samples can include, but are notlimited to, blood, urine, fine needle aspirates, pancreatic juice, orany other tissue, including, but not limited to, a biopsy from thepancreas or surrounding tissue. The tissue sample can be any type ofroutine pathologically prepared sample including any type of tissueaffixed to a microscope slide, plate or well. In a preferred embodiment,the tissue sample is a frozen section of tissue. Pancreatic juice can beobtained from patients undergoing diagnostic ERCP procedure. It can alsobe collected non-invasively from asymptomatic individuals or inhigh-risk populations by secretin stimulation and sampling of pancreaticjuice using duodenoscope. Fine needle biopsy samples from pancreaticcancer patients or pancreatic tumor tissues can be collected aftersurgery if it is a resectable tumor.

Analysis: The inventors contemplate that results obtained from one typeof sample collection can be compared with those obtained from another inorder to aid in the identification, monitoring or detection of a tumormarker or in the diagnosis of, or monitoring the progression of thecancer. Subsequently, the same slides can be stained and analyzed by acytopathologist for the presence of benign, atypical or malignant cells.Such staining can include routing cytological stains such as H&E orimmuostaining with specific antibodies.

The sensitivity of the MB-based detection using blood and pancreas juiceis evaluated by comparing the size and stages of the pancreatic cancerlesions diagnosed by imaging technologies such as helical CT, MRI orendoscopic ultrasound or pathological diagnosis after surgical resectionof the cancer.

Monitoring the Presence of a Mutant Gene

The present invention also provides methods for detecting the presenceof a mutant gene in a tumor cell using the molecular beacon technologydisclosed herein. It is contemplated that the monitoring for thepresence of such mutated genes such as the K-ras gene, will aid in thediagnosis and treatment of various types of cancers, including but notlimited to pancreatic cancer.

Monitoring Alterations in Gene Expression

A method of monitoring alterations in gene expression in viable cells inreal time using the molecular beacon technology disclosed herein. Suchmethods will allow for the monitoring expression of target genesincluding, but not limited to, tumor markers, mutant genes or the like.Thus, clinicians will be able to detect and monitor the development ofcancers in for example, individuals who have been determined to begenetically predisposed to certain cancers. In this way, propertreatments can be implemented early in the course of the development ofthe disease, which may indeed prevent or diminish the onset of tumor orcancer growth. Importantly, detection of the level of gene expression inviable cells will allow one to measure the changes of gene expressionreal-time in the same cell population after various treatments. Thisapproach can be used for the examination of alternation of geneexpression in tumor cells by biological reagents as well as for theevaluation of the expression of molecular target genes after treatmentof the cancer cells with therapeutic reagents.

A diagnostic kit for detecting alterations in gene expression in viablecells in real-time is also contemplated that would include any materialsor reagents suitable for carrying out the disclosed methods.

Detection of Cancerous Cells

The present invention provides a method of detecting cancerous cells ina sample using the molecular beacon technology disclosed herein. It iscontemplated that cancer cells can be detected that originate from oneor more of the cancers including but not limited to, breast, pancreas,ovarian, prostate, colorectal, hepatocellular, multiple myeloma,lymphoma, bladder, medullary carcinoma of the thyroid, neuroendocrinetumors, carcinoid tumors, testicular, gestational trophoblast neoplasms,lung, melanoma and stomach. A diagnostic kit for detecting cancerouscells is also contemplated that would include any materials or reagentssuitable for carrying out the disclosed methods.

Without intent to limit the scope of the invention, exemplary methodsand their related results according to the embodiments of the presentinvention are given below. Note that titles or subtitles may be used inthe examples for convenience of a reader, which in no way should limitthe scope of the invention. Moreover, certain theories are proposed anddisclosed herein; however, in no way they, whether they are right orwrong, should limit the scope of the invention so long as data areprocessed, sampled, converted, or the like according to the inventionwithout regard for any particular theory or scheme of action.

EXAMPLES Example 1

Preliminary Detection of Survivin Gene Expression in Breast Cancer CellLines Using Survivin MB

MBs specific for human survivin gene were designed and synthesized, withthe probe sequence complementary to the cDNA sequence between 27 nt to43 nt of the gene (5′-Alexa-fluo 488-CTGAGAAAGGGCTGCCAGTCTCAG-Dabcyl-3′;SEQ ID NO:1). The underlined stem sequences of survivin MB is speciallydesigned to achieve the best thermodynamic effect. At first, specifichybridization of survivin MB was studied with a synthesized survivinoligonucleotide target in vitro.

Results indicated that the survivin MB binds specifically to survivintargets (FIG. 2 C). The specificity of survivin MBs for detectingsurvivin mRNA was further examined in the breast cancer cell linesMDA-MB-231, MDA-MB-435 and MCF-7 expressing different levels of survivingene and in normal human mammary epithelial cell line (MCF-10A). Afterincubation of survivin MBs with fixed cells at 37° C. for 1 hour, thecells were washed with PBS and observed under Nikon fluorescencemicroscope. Cells were grown on chamber slides and fixed with acetone;survivin MB was labeled with Alexa-Fluo 488 (Green, FIG. 4 A). Thedetection of survivin expression in breast cancer and normal cell linesby Western blot is shown in FIG. 4 B. As shown in FIG. 4 A, tumor cellsdisplay from intermediate to strong red fluorescence while in MCF-10Acells, only a weak fluorescence was observed. Furthermore, the intensityof the fluorescence is correlated well with the level of survivindetected by Western blots (FIG. 4 B).

Example 2

Simultaneous Detection of Expression of Survivin and Cyclin D 1 inBreast Cancer Cells

Further studies were carried out to examine the specificity of detectionof cancer cells with several tumor marker MBs. MBs for cyclin D 1 andHer-2/neu genes were designed and synthesized (Table 1). Inventors thenexamined the specificity of the MBs in vitro with synthesizedolignucleotide targets. The sequences for each MB are shown in theTable 1. The underlined sequences are not part of the gene and aredesigned to form the stem. Inventors have also synthesized a severalsurvivin MB with the same sequences as with different fluorescence dyes(6-FAM, Cy3, Alexa-Fluo-488). Survivin MB-FITC has a different targetsequence as shown in the Table 2. The MBs were synthesized by MWGBiotech (High point, N.C.) and Integrated DNA Technologies, Inc. (IDT,Coralville, Iowa).

To determine specific hybridization of the MBs with their targets,hybridization studies were carried out by mixing the MBs with theiroligonucleotide targets and then the fluorescence intensities weremeasured by a fluorescence microplate reader. Control groups for thisstudy were the MBs mixed with oligonucleotide targets from a differentgene (FIGS. 2C and D). After demonstration of specific binding of theMBs with their targets, the specificity of the MBs in detection ofcyclin D 1 in human breast tumor and normal cell lines was furtherexamined (FIG. 4). TABLE 1 Molecular beacons for detection of thesurvivin, cyclin D 1 and Her-2/neu mRNA Excitation and Emission offluorescence dye Genes MB design (Color) Survivin Survivin mRNA (27 to43 nt) Ex 491 nm 5′-Alexa Fluo 488- Em 515 nm (Green)CTGAGAAAGGGCTGCCAGTCTCAG-Dabcyl-3′ (SEQ ID NO:2) Cyclin D 1 Cyclin D 1MB1 mRNA (376-394 nt) Ex 596 nm 5′-Texas red-TGGAGTTGTCGGTGTAGACTCCA- Em615 nm (red) Dabcyl-3′-(SEQ ID NO: 3) Cyclin D 1 MB2 mRNA (698-715 nt)5′-Texas red-CACTTGATCACTCTGGACAAGTG- Dabcyl-3′-(SEQ ID NO: 4) Her-2/neuHer-2/neu MB1 mRNA (Exon 2) Ex 346 nm 5′-Alexa Fluo350-TAGAGGTGGCGGAGCATCTCTA- Em 442 nm (Blue) Dabcyl-3′-(SEQ ID NO: 5)Her-2/neu MB2 mRNA (Exon 3) 5′-Alexa Fluo 350-CAATCCGCAGCCTCTGCGATTG-Dabcyl-3′-(SEQ ID NO:6)

For detection of breast cancer cells with cyclin D 1 and survivin MBs, amixture of 100 nM of the cyclin D1 and survivin MBs were incubated withacetone-fixed MDA-MB-231, SKBr3, MDA-MB-435, MCF-7 or MCF-10 A cells for1 hr. The fluorescence intensity was examined under a fluorescencemicroscope (FIG. 4A). Levels of survivin and cyclin D1 proteins wereexamined by Western blot analysis are shown in FIG. 4 B, which indicateda correction between the level of survivin and cyclin D1 mRNA detectedby the MBs with the protein levels (FIG. 4B). FIG. 7 shows the detectionof gene expression in real time in living cells using molecular beaconsfollowing treatment of human breast cell cancer line MCF-7 cells withEGF. FIG. 8 showed that treatment of docetaxel increased the level ofsurvivin gene expression in breast cancer cell lines.

Example 3

Identification of Survivin Expressing Cells in Primary Breast CancerTissues

To investigate the feasibility of using survivin as a breast cancermarker, the expression of survivin proteins in paired and non-pairednormal and breast cancer tissues by Western blot analysis was examined.It was found that that survivin is expressed in over 73% of breastcancer tissues but not in any of the normal breast tissues FIG. 10).Immunofluorescence staining of frozen tissue sections with survivinantibody also revealed that survivin is highly expressed in invasiveductal carcinoma cells but not in normal breast ductal cells (FIG. 5A).Interestingly, the lesions of DCIS also showed intermediate level ofsurvivin (FIG. 5A). Examination of survivin gene expression on frozentissue sections using survivin MBs further revealed survivin-expressingcancer cells in DCIS lesion, invasive ductal carcinoma, and metastasesin draining lymph node of a breast cancer patient. However, there wereno survivin positive cells found in normal breast tissues (FIGS. 5A andB).

Example 4

Design of MBs for Detection of Mutant K-ras and Survivin mRNA inPancreatic Cancer Cell and Tissues

Having determined the parameters for beacon structures and hybridizationconditions, MBs for detecting the expression of K-ras mutation geneswere designed and synthesized. These are depicted in Table 4. Since over80% of K-ras mutations are found in K-ras codon 12 (Vos et al., J. ofPath. 187(3):279-84 (1999)), K-ras MB 1 that detect GGT-GAT (Gly to Asp)transition and K-ras MB2, targeting GGT to GTT mutation, weresynthesized (Table 2). GGT to GAT is one of the most common K-ras pointmutations in pancreatic cancer (Table 3).

K-ras MB 1 or MB2 selectively binds to its DNA target in vitro andproduces stronger fluorescence signal as compared with othernon-specific DNA targets (FIG. 2). Examination of specificity of K-rasMBs in pancreatic cancer cell lines also demonstrated that K-ras MBs candetect pancreatic cancer cells with a specific k-ras mutation. As shownin the FIG. 3, Panc-1 cell line has a K-ras GGT to GAT mutation andshowed strong fluorescence intensity after incubating with K-ras MB 1,but displayed a weak fluorescence in K-ras MB2-stained cells. Capan-2cell line contains a K-ras GGT to GTT mutation and a brighterfluorescence was detected in K-ras MB 2 stained cells. Both cell linesexpressed a high level of survivin as detected by survivin MB (FIG. 3A).On the other hand, K-ras MBs did not produce strong fluorescencesignaling in MIA PaCa-2 cell line, which has a K-ras GGT to TGTmutation, or K-ras wild type BXPC-3 cells. However, those cells werepositive for survivin MB staining. Importantly, incubation of K-ras andsurvivin MBs did not produce detectable fluorescence signaling in anormal cell line (HDF), which is generated from normal dermalfibroblasts (FIG. 3B).

K-ras MBs were also able to detect pancreatic cancer cells with K-rasmutations on Frozen tissue sections. After incubating with K-ras MB 1 orK-ras MB 2 for 1 hour and counterstained with Hoechst 33342 (blue), Theslides were observed under fluorescence microscope with a digitalimaging system. K-ras MB 1 detected the cancer cells expressing a GGT toGAT mutant K-ras gene on the frozen sections of pancreatic cancertissues from patient #1 and #2, which contained K-ras codon 12 GGT toGAT mutation. However, bright red fluorescent cells were found on frozensections of pancreatic cancer tissues from patient #5, which had a K-rasGGT to GTT mutation after incubation with K-ras MB 2 but not with K-rasMB1 (FIG. 6A).

The feasibility of detection of survivin expression in pancreatic cancertissues by survivin MB was also examined. At first, expression ofsurvivin in the pancreatic cancer tissues was demonstrated byimmunofluorescence staining with an anti-survivin monoclonal antibody.Inventors showed that survivin protein was highly expressed inpancreatic cancer tissues but was undetectable in normal pancreas (FIG.6B). TABLE 2 Design of molecular beacons for K-ras codon 12 mutation andsurvivin genes and corresponding target sequences A. Design of the MBsfor detecting mutant K-ras and survivin mRNAs MBs Target sequencesDesign of the MBs K-ras MB 1 K-ras codon 12 GGT5′-Cy3-CCTACGCCATCAGCTCCGTAGG-Dabcyl-3′ to GAT mutation (SEQ ID NO 7)K-ras MB 2 K-ras codon 12 GGT5′-Texas-red-CCTACGCCAACAGCTCCGTAGG-Dabcyl-3′ to GTT mutation (SEQ ID NO8) Survivin MB 1 Survivin cDNA from 5′-Cy3 (Alexia-Fluo488 or 6-FAM)- 27to 43 nucleotide CTGAGAAAGGGCTGCCAGTCTCAG-Dabcyl-3′ (SEQ ID NO 2)Survivin MB 2 Survivin cDNA from5′FITC-TGGTCCTTGAGAAAGGGCGACCA-Dabcyl-3′ 32 to 51 nucleotide (SEQ ID NO9) GAPDH MB GAPDH cDNA from 5′-Cy3-GAGTCCTTCCACGATACCGACTC-Dabcyl-3′ 504to 521 nucleotide (SEQ ID NO 10) B. Oligonucleotides served as DNAtargets for K-ras and survivin MBs DNA Targets SynthesizedOligonucleotide sequences K-ras wild type 5′-GTA GTT GGA GCT GGT GGC GTAGGC AAG AGTGCCTTGACGATACAGCTAATT CAG-3′ (GGT) K-ras Mut 1 (GAT) 5′-GTAGTT GGA GCT GAT GGC GTA GGC AAG AGTGCCTTGACGATACAGCTAATT CAG-3′ K-rasMut 2 (GTT) 5′-GTA GTT GGA GCT GTT GGC GTA GGCAAGAGTGCCTTGACGATACAGCTAATT CAG-3′ Survivin5′-CCTGCCTGGCAGCCCTTTCTCAAGGACCACCGCATCTCTACATTCAAGAAC-3′

TABLE 3 K-ras mutations in pancreatic cancer cell lines Percentage ofthe Mutation in Primary K-ras Predicated Pancreatic Cell lines mutationAlteration Products Tissues Panc-1 Yes, GGT to GAT GLY to ASP 48+/101(48%) Codon 12 Capan-2 Yes, GGT to GTT GLY to VAL 33+/101 (33%) Codon 12Miapaca-2 Yes, GGT to TGT GLY to CYS  2+/101 (2%) Codon 12 PSN-1 Yes,GCT to CGT GLY to ARG  9+/101 (9%) Codon 12 BXPC-3 No No GLY No HDF NoNo GLY No

TABLE 4 Design of molecular beacons targeting K-ras codon 12 mutations5′-ATG ACT GAA TAT AAA CTT GTG GTA GTT GGA GCT GGT GGC GTA GGC AAG AGTGCC TTG ACG-3′ Molecular Beacons GGT-GAT: 5′-Cy 3- (SEQ ID NO: 7)CCTACGCCATCAGCTCCGTAGG-Dabcyl-3′- GGT-GTT: 5′-Cy-3- (SEQ ID NO: 8)CCTACGCCAACAGCTCCGTAGG-Dabcyl-3′- GGT-CGT: 5′-Cy-3- (SEQ ID NO: 11)CCTACGCCACGAGCTCCGTAGG-Dabcyl-3′- GGT-TGT: 5′-Cy-3- (SEQ ID NO: 12)CCTACGCCACAAGCTCCGTAGG-Dabcyl-3′- GGT-GCT: 5′-Cy-3- (SEQ ID NO: 13)CCTACGCCAGCAGCTCCGTAGG-Dabcyl-3′-Codon 12 = underlined

The above specification, examples, and data provide a completedescription of the manufacture and use of the invention. Unlessotherwise specified, all patent and non-patent references cited arehereby incorporated by reference for background information only. Sincemany embodiments of the invention can be made without departing from thespirit and scope of the invention, the invention resides in the claimshereinafter appended.

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1. A method of detecting the presence of at least one tumor marker mRNAin a sample comprising: i) providing a sample of cells for analysis; ii)treating the sample with an oligonucleotide that targets the tumormarker mRNA, wherein the oligonucleotide comprises at least one linkedenergy donor moiety and at least one linked energy acceptor moiety,wherein said oligonucleotide forms a stem-loop hairpin and wherein saiddonor and acceptor moieties are separated by at least a portion of aprobing nucleobase sequence; iii) detecting, identifying or quantitatingthe hybridization of the target sequence under suitable hybridizationconditions, wherein the presence, absence or amount of target sequencepresent in the sample is correlated with a change in detectable signalassociated with at least one donor or acceptor moiety of theoligonucleotide; and iv) detecting, identifying or quantitating thepresence of a tumor marker based upon the presence, absence or amount ofthe hybridization of the oligonucleotide to the target sequence that isdetermined.
 2. The method of claim 1, wherein the tumor marker is one ormore of the markers selected from the group consisting of survivin,cyclin D1, Her2/neu, a mutant K-ras, chymotrypsinogen, basic fiborblastgrowth factor, carcinoembryonic antigen, prostate, specific antigen,alpha-fetalprotein, beta-2-microglobulin, bladder tumor antigen,chromogranin A, neuron-specific enolase, S-100, TA-90, tissuepolypeptide antigen and human chorionic gonadotropin
 3. The method ofclaim 1, wherein the sample taken from at least one source selected fromthe group consisting of blood, urine, pancreatic juice, ascites, breastductal lavage, nipple aspiration, needle biopsy or tissue.
 4. The methodof claim 3, wherein the tissue is a biopsy from the pancreas or breast.5. The method of claim 3, wherein the tissue is a frozen section.
 6. Themethod of claim 1, wherein the sample is taken from a breast ductallavage.
 7. The method of claim 1, wherein the sample is taken frompancreatic juice.
 8. The method of claim 1, wherein the quantificationof the presence of the tumor marker is accomplished by FACS-scananalysis.
 9. The method of claim 1, wherein the olignucleotide is one ormore selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12 and
 13. 10. The method of claim 1, wherein theoligonucleotide targets the tumor marker survivin.
 11. The method ofclaim 10, wherein the oligonucleotide is one or more selected from thegroup consisting of SEQ ID NOS: 1, 2 and
 9. 12. The method of claim 1,wherein the oligonucleotide targets the tumor marker cyclin D1.
 13. Themethod of claim 12, wherein the oligonucleotide is one or more selectedfrom the group consisting of SEQ ID NOS: 3 and
 4. 14. The method ofclaim 1, wherein the oligonucleotide targets the tumor marker Her2/neu.15. The method of claim 14, wherein the oligonucleotide is one or moreselected from the group consisting of SEQ ID NOS: 5 and
 6. 16. Themethod of claim 1, wherein the oligonucleotide targets the tumor markera K-ras mutant gene.
 17. The method of claim 16, wherein theoligonucleotide is one or more selected from the group consisting of SEQID NOS: 7, 8, 11, 12 and
 13. 18. A method of detecting the presence of amutant gene in a tumor cell comprising: i) providing a sample of tumorcells for analysis; ii) treating the sample with an oligonucleotide thattargets the mutant gene, wherein the oligonucleotide comprises at leastone linked energy donor moiety and at least one linked energy acceptormoiety, wherein said oligonucleotide forms a stem-loop hairpin andwherein said donor and acceptor moieties are separated by at least aportion of a probing nucleobase sequence; iii) detecting, identifying orquantitating the hybridization of the oligonucleotide to the mutant genetarget sequence under suitable hybridization conditions, wherein thepresence, absence or amount of mutant gene target sequence present inthe sample is correlated with a change in detectable signal associatedwith at least one donor or acceptor moiety of the oligonucleotide; andiv) detecting, identifying or quantitating the presence of a mutant genebased upon the presence, absence or amount of the hybridization of theoligonucleotide to the mutant gene target sequence that is determined.19. The method of claim 18, wherein the mutant gene is a mutant K-rasgene.
 20. The method of claim 19, wherein the quantification of thepresence of the mutant gene is accomplished by FACS-scan analysis. 21.The method of claim 19, wherein the oligonucleotide is one or moreselected from the group consisting of SEQ ID NOS: 7, 8, 11, 12, and 13.22. A method of monitoring alterations in gene expression in viablecells comprising: i) providing a sample of viable cells for analysis;ii) treating the sample with an oligonucleotide that targets aparticular gene sequence, wherein the oligonucleotide comprises at leastone linked energy donor moiety and at least one linked energy acceptormoiety, wherein said oligonucleotide forms a stem-loop hairpin andwherein said donor and acceptor moieties are separated by at least aportion of a probing nucleobase sequence; iii) detecting, identifying orquantitating the hybridization of the target sequence under suitablehybridization conditions, wherein the presence, absence or amount oftarget sequence present in the sample is correlated with a change indetectable signal associated with at least one donor or acceptor moietyof the oligonucleotide; and iv) detecting, identifying or quantitatingthe alteration in the expression of the particular gene based upon thepresence, absence or amount of the hybridization of the oligonucleotideto the target sequence that is determined.
 23. The method of claim 22,wherein the quantification of the alteration in gene expression isaccomplished by FACScan analysis and by a fluorescence microplatereader.
 24. A method of detecting or monitoring presence or progressionof breast cancer in a subject comprising: i) providing a sample of cellsfrom said subject for analysis; ii) treating the sample with anoligonucleotide that targets a breast cancer marker gene sequence,wherein the oligonucleotide comprises at least one linked energy donormoiety and at least one linked energy acceptor moiety, wherein saidoligonucleotide forms a stem-loop hairpin and wherein said donor andacceptor moieties are separated by at least a portion of a probingnucleobase sequence; iii) detecting, identifying or quantitating thehybridization of the target oligonucleotide sequence to the breastcancer marker gene sequence under suitable hybridization conditions,wherein the presence, absence or amount of target oligonucleotidesequence present in the sample is correlated with a change in detectablesignal associated with at least one donor or acceptor moiety of theoligonucleotide; and iv) detecting, identifying or quantitating thepresence or progression of breast cancer based upon the presence,absence or amount of the hybridization of the oligonucleotide to thebreast cancer target sequence that is determined.
 25. The method ofclaim 24, wherein the breast cancer marker is one or more of the markersselected from the group consisting of survivin, cyclin D1, Her2/neu,basic fiborblast growth factor, EGF receptor and carcinoembryonicantigen.
 26. The method of claim 24, wherein the sample is taken from atleast one source selected from the group consisting of blood, urine,breast ductal lavage, ascites, nipple aspiration, needle biopsy ortissue.
 27. The method of claim 26, wherein the tissue is a biopsy froma breast or lymph node.
 28. The method of claim 26, wherein the tissueis a frozen section.
 29. The method of claim 24, wherein thequantification of the presence or progression of the breast cancer isaccomplished by FACS-scan analysis.
 30. The method of claim 24, whereinthe olignucleotide targets the breast cancer marker survivin.
 31. Themethod of claim 30, wherein the oligonucleotide is one or more selectedfrom the group consisting of SEQ ID NOS: 1, 2 and
 9. 32. The method ofclaim 24, wherein the oligonucleotide targets the breast cancer markercyclin D1.
 33. The method of claim 32, wherein the oligonucleotide isone or more selected from the group consisting of SEQ ID NOS: 3 and 4.34. The method of claim 24, wherein the oligonucleotide targets thebreast cancer marker Her2/neu.
 35. The method of claim 34, wherein theoligonucleotide is one or more selected from the group consisting of SEQID NOS: 5 and
 6. 36. A method of detecting or monitoring presence orprogression of pancreatic cancer in a subject comprising: i) providing asample of cells from said subject for analysis; ii) treating the samplewith an oligonucleotide that targets a pancreatic cancer marker genesequence, wherein the oligonucleotide comprises at least one linkedenergy donor moiety and at least one linked energy acceptor moiety,wherein said oligonucleotide forms a stem-loop hairpin and wherein saiddonor and acceptor moieties are separated by at least a portion of aprobing nucleobase sequence; iii) detecting, identifying or quantitatingthe hybridization of the target oligonucleotide sequence to thepancreatic cancer marker gene sequence under suitable hybridizationconditions, wherein the presence, absence or amount of targetoligonucleotide sequence present in the sample is correlated with achange in detectable signal associated with at least one donor oracceptor moiety of the oligonucleotide; and iv) detecting, identifyingor quantitating the presence or progression of pancreatic cancer basedupon the presence, absence or amount of the hybridization of theoligonucleotide to the breast cancer target sequence that is determined.37. The method of claim 36, wherein the pancreatic cancer marker is oneor more of the markers selected from the group consisting of survivin, amutant K-ras gene, and carcinoembryonic antigen.
 38. The method of claim36, wherein the sample is taken from at least one source selected fromthe group consisting of blood, urine, pancreatic juice, ascites, needlebiopsy or tissue.
 39. The method of claim 38, wherein the tissue is afrozen section.
 40. The method of claim 36, wherein the quantificationof the presence or progression of the pancreatic cancer is accomplishedby FACS-scan analysis.
 41. The method of claim 36, wherein theolignucleotide targets the pancreatic cancer marker survivin.
 42. Themethod of claim 41, wherein the oligonucleotide is one or more selectedfrom the group consisting of SEQ ID NOS: 1, 2 and
 9. 43. The method ofclaim 36, wherein the oligonucleotide targets a mutant K-ras pancreaticcancer marker.
 44. The method of claim 43, wherein the oligonucleotideis one or more selected from the group consisting of SEQ ID NOS: 7, 8,11, 12 and
 13. 45. A method of detecting cancerous cells in a samplecomprising: i) providing a sample of cells for analysis; ii) treatingthe sample with an oligonucleotide that targets a cancer-specific markergene sequence, wherein the oligonucleotide comprises at least one linkedenergy donor moiety and at least one linked energy acceptor moiety,wherein said oligonucleotide forms a stem-loop hairpin and wherein saiddonor and acceptor moieties are separated by at least a portion of aprobing nucleobase sequence; iii) detecting, identifying or quantitatingthe hybridization of the target oligonucleotide sequence to thepancreatic cancer marker gene sequence under suitable hybridizationconditions, wherein the presence, absence or amount of targetoligonucleotide sequence present in the sample is correlated with achange in detectable signal associated with at least one donor oracceptor moiety of the oligonucleotide; and iv) detecting, identifyingor quantitating the presence cancerous cells based upon the presence,absence or amount of the hybridization of the oligonucleotide to thecancer-specific target sequence that is determined.
 46. The method ofclaim 45, wherein the quantification of the presence of a cancer cell isaccomplished by FACS-scan analysis.
 47. The method of claim 45, whereinthe cancer cell originates from one or more of the cancers selected fromthe group consisting of: breast, pancreas, ovarian, prostate,colorectal, hepatocellular, multiple myeloma, lymphoma, bladder,medullary carcinoma of the thyroid, neuroendocrine tumors, carcinoidtumors, testicular, gestational trophoblast neoplasms, lung, melanomaand stomach.
 48. A diagnostic kit for detecting or monitoring theprogession of cancerous cells comprising materials suitable for carryingout the method of claim
 36. 49. A diagnostic kit for detectingalterations in gene expression in viable cells in real-time comprisingmaterials suitable for carrying out the method of claim 22.